Fluorescent protein development 1) We have an ongoing project to develop improved green photoactivatable fluorescent proteins (PAFPs) for Photoactivated Localization Microscopy (PALM). Currently, PAFPs are available in essentially two colors, green and red. Red PAFPs have proven to be useful for PALM by providing low backgrounds and sufficient numbers of photons capable of <25 nm uncertainty in localization. Green PAFPs suffer from low photons and high backgrounds, which limit precise molecular localization. We are currently working on developing an improved green PAFP for PALM. Previous improvements included variants of PAGFP with low background, but at the expense of photoactivation contrast. Efforts are aimed at reengineering by site-directed mutagenesis high photoactivation contrast while maintaining the low background fluorescence. 2) A project related to PAFPs as well as conventional fluorescent proteins, whether current or under development, is the characterization of their blinking behaviors. Ideally, PAFPs would be turned on, fluoresce for a given period of time, photobleach, and never turn on again. However, common to almost all fluorophores, PAFPs sample dark states during which they do not produce photons. We have devised assays and analyses to survey existing molecules in efforts to determine parameters that can correctly identify blinking molecules, combine their collected photons and correctly localize them only once. In addition, we are testing variants with mutations within and around the PAFP chromophores to determine which affect the blinking on times, blinking off times, and percentage of blinking molecules in a population. 3) An offshoot of the previous project is a study of the little known green-to-red photoconversion which occurs in the commonly used EGFP variant. Our own studies have found that EGFP can undergo a green-to-red photoconversion in aerated samples lacking oxidizing agents, suggesting it is an inherent property of the protein. A survey of FP variants has found one which does not undergo this process. Comparison of the primary structures shows several differences and by performing single point mutagenesis at those positions the green-to-red photoconversion has been recovered. Now we are targeting the amino acid position for mutagenesis in EGFP of interest to both inhibit photoconversion as well as enhance this phenomenon. As second part of this project is to analyze the photobleaching behavior (loss in green fluorescence) in concert with redding effect. 4) We collaborate with Joy Zhao and Peter Schuck on their development of new fluorescence ultracentrifugation techniques and oligomerization characteristics of fluorescent proteins. Cell biology projects 1) We are collaborating with Moshe Levi (UCHSC) on a project to image with PALM the localization of sodium phosphate transporters, NaPi-2a and NaPi-2c, in the apical brush border membrane (BMM) of opossum kidney (OK) cells. These molecules play key roles in renal proximal tubule inorganic phosphate (Pi) reabsorption and help maintain Pi homeostasis. These transporters respond to increases and decreases in dietary Pi by changing their abundance in the BMM via trafficking to and from the BMM and an intracellular compartment. The transporters are known to interact with a number of PDZ containing proteins, such as NHERF-1 and NHERF-3, and to be associated with microdomains enriched in cholesterol and glycosphingolipids. 2) We collaborate with Anamaris Colberg-Poley on super-resolution imaging of human cytomegalovirus infected cells. Our interest is gaining insight into the transfer of pUL37x1 protein from mitochondria associated membranes to the outer mitochondria membrane. 3) With Raul Rojas, a staff scientist in the lab of Lawrence Tabak, we have undertaken a project to image with PALM the localization of Golgi apparatus enzymes. These studies are intended to help in our understanding of where the enzymes are located within the Golgi and what role these locations may play in the enzymatic activity. Specifically, we are interested in whether enzymes involved in early steps in sugar modifications of proteins are located in early compartments of the Golgi and vice versa. Instrumentation and imaging development 1) The major instrument in the lab consists of an Olympus IX-71. In collaboration with Hari Shroffs group (NIBIB), two-photon excitation via temporal focusing and/or multi-focal excitation has been implemented to allow single plane photoactivation and PALM studies further away from the coverglass than allowed by TIRF microscopy. A cylindrical lens inserted into the emission pathway to defocus the fluorescence signals from single molecules. This allows localization of the molecules in the axial direction in addition to the normal x, y directions. 2) Two-photon excitation on our PALM system has been upgraded using a microlens array to produce a grid pattern of diffraction limited spots. This upgrade allows us to implement a new super-resolution structured illumination microscopy (SIM) technique, multi-focal SIM, developed by the Shroff lab. This instrument represents the next phase in MSIM development by incorporating multi-photon excitation. This allows super-resolution imaging in tissue at depths unattainable by current super-resolution techniques. 3) A total internal reflection fluorescence (TIRF) microscope system has been built by Yan Fu on an Olympus IX70 microscope obtained through property transfer. This instrument is been designed to provide a more homogeneous illumination pattern compared with traditional through objective TIRF by illuminating the sample from all angles possible for a through objective configuration. Currently, Yan is comparing the scanning TIRF configuration with the traditional single angle TIRF, testing our newly devised method to measure TIRF depth, and refining a new technique for building and aligning TIRF microscopes. We have also developed a method to image at multiple positions with the TIRF excitation zone which effectively allows optical sections at 20-50 nm increment through the 200-300 nm illumination region. 4) We collaborate with the Andy York and Hari Shroff on the development of a new image processing technique relying on joint deconvolution of multiple images with various strengths which complement and overcome their various weaknesses. With this method, images with high signal-to-noise but low resolution are deconvolved jointly with images of low signal but high resolution to produce an image with high S/N and high resolution.